The present invention relates to the field of molecular biology, especially as related to methods for sensitive assays for assessing protein-protein, protein-ligand or protein-nucleic acid interactions, and antagonists and/or agonists of such interactions. Specifically this invention relates to split monomeric reporter protein systems including, but not limited to, split luciferase, β-lactamase or fluorescent protein reporter systems, and excluding beta-galactosidase, where a functional protein results when the portions interact, with the result that there is a detectable signal produced in the assay. In particular, the split reporter is expressed in a cell-free system.
Protein-protein (1) and protein-nucleic acid (2) interactions are central to cellular function and are also emerging targets for pharmacological intervention when implicated in a particular disease pathway. Thus numerous in vitro and in vivo methods have been developed to target (3-7) and study these biomolecular interactions. Widely utilized in vitro methods for interrogating protein-protein and protein-DNA interactions and their antagonists include variations of enzyme linked immunosorbent assays (ELISAs), surface plasmon resonance (SPR), fluorescence resonance energy transfer (FRET) and fluorescence polarization (FP), which either require the use of antibodies or purified proteins and in some cases require chemical derivatization. On the other hand, powerful in vivo methods such as the yeast two-hybrid (8) assays have the advantage of speed by eliminating the need for protein purification but can be subject to false positives and negatives due to the multifactorial nature of signal generation (9). In between these two extremes lies the protein fragment based methods, where a specific biomolecular interaction drives the reassembly of a previously split reporter protein (10) (FIG. 1).
Whereas there are various methods employing split reporter proteins, the present inventors are not aware of any methods in which there is cell-free expression of one or both of the split monomeric reporter proteins and subsequent assay of the expressed, assembled reporter in such an assay. Examples of methods employing living cells or transgenic organisms are provided in US Patent Publications 2005/0144661, 2004/0235064; 2007/0161067; 2006/0224331; and U.S. Pat. Nos. 6,897,017; 6,872,871; 7,166,424; 7,160,691; 6,828,099; 6,428,951; 6,929,916; 7,062,219; and 7,176,287. See also Kim et al. (130); Porter et al. (23); Porter et al. (58); Paulmurugan et al. (131).
There is a need in the art for assays of molecular interactions which are fast, require relatively little culture and handling of samples, and are sensitive, accurate and precise.